Résumé:
« Chitin and peptidoglycan fragments are well recognized as pathogen associated molecular patterns (PAMPs). Long-chain oligosaccharides of (1→4)-linked N-acetyl-D-glucosamine (GlcNAc) units indeed activate plants and mammals innate immune system. However, the mechanisms underlying PAMPs perception by lysine motif (LysM) domain receptors remain largely unknown because of insufficient availability of high-affinity molecular probes. Here, we report a two-enzyme cascade to synthesize long-chain (1→4)-linked GlcNAc oligomers. Expression of the D52S mutant of hen egg-white lysozyme (HEWL) in Pichia pastoris at 52 mg.L-1 provided a new glycosynthase catalyzing efficient polymerization of -chitintriosyl fluoride. Selective N-deacetylation at the non-reducing unit of the glycosyl fluoride donor by Sinorhizobium meliloti NodB chitin-N-deacetylase abolished its ability to be polymerized by the glycosynthase but not to be transferred onto an acceptor. Using NodB and D52S HEWL in
a one-pot cascade reaction allowed the synthesis on a milligram scale of chitin hexa-, hepta- and octasaccharides with yields up to 65% and a perfect control over their size. »
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